Efficient Enhancement of Extracellular Electron Transfer in Shewanella oneidensis MR-1 via CRISPR-Mediated Transposase Technology.

Electroactive bacteria, exemplified by Shewanella oneidensis MR-1, have garnered significant attention due to their unique extracellular electron-transfer (EET) capabilities, which are crucial for energy recovery and pollutant conversion. However, the practical application of MR-1 is constrained by its EET efficiency, a key limiting factor, due to the complexity of research methodologies and the challenges associated with the practical use of gene editing tools. To address this challenge, a novel gene integration system, INTEGRATE, was developed, utilizing CRISPR-mediated transposase technologies for precise genomic insertion within the S. oneidensis MR-1 genome. This system facilitated the insertion of extensive gene segments at different sites of the Shewanella genome with an efficiency approaching 100%. The inserted cargo genes could be kept stable on the genome after continuous cultivation. The enhancement of the organism's EET efficiency was realized through two primary strategies: the integration of the phenazine-1-carboxylic acid synthesis gene cluster to augment EET efficiency and the targeted disruption of the SO3350 gene to promote anodic biofilm development. Collectively, our findings highlight the potential of utilizing the INTEGRATE system for strategic genomic alterations, presenting a synergistic approach to augment the functionality of electroactive bacteria within bioelectrochemical systems.

Optimized Antibiotic Resistance Genes Monitoring Scenarios Promote Sustainability of Urban Water Cycle.

Dissemination of antibiotic resistance genes (ARGs) in urban water bodies has become a significant environmental and health concern. Many approaches based on real-time quantitative PCR (qPCR) have been developed to offer rapid and highly specific detection of ARGs in water environments, but the complicated and time-consuming procedures have hindered their widespread use. Herein, we developed a facile one-step approach for rapid detection of ARGs by leveraging the trans-cleavage activity of Cas12a and recombinase polymerase amplification (RPA). This efficient method matches the sensitivity and specificity of qPCR and requires no complex equipment. The results show a strong correlation between the prevalence of four ARG markers (ARGs: sul1, qnrA-1, mcr-1, and class 1 integrons: intl1) in tap water, human urine, farm wastewater, hospital wastewater, municipal wastewater treatment plants (WWTPs), and proximate natural aquatic ecosystems, indicating the circulation of ARGs within the urban water cycle. Through monitoring the ARG markers in 18 WWTPs in 9 cities across China during both peak and declining stages of the COVID epidemic, we found an increased detection frequency of mcr-1 and qnrA-1 in wastewater during peak periods. The ARG detection method developed in this work may offer a useful tool for promoting a sustainable urban water cycle.

Direct ammonia oxidation (Dirammox) is favored over cell growth in Alcaligenes ammonioxydans HO-1 to deal with the toxicity of ammonium.

Bacteria capable of direct ammonia oxidation (Dirammox) play important roles in global nitrogen cycling and nutrient removal from wastewater. Dirammox process, NH3 → NH2 OH → N2 , first defined in Alcaligenes ammonioxydans HO-1 and encoded by dnf gene cluster, has been found to widely exist in aquatic environments. However, because of multidrug resistance in Alcaligenes species, the key genes involved in the Dirammox pathway and the interaction between Dirammox process and the physiological state of Alcaligenes species remain unclear. In this work, ammonia removal via the redistribution of nitrogen between Dirammox and microbial growth in A. ammonioxydans HO-1, a model organism of Alcaligenes species, was investigated. The dnfA, dnfB, dnfC, and dnfR genes were found to play important roles in the Dirammox process in A. ammonioxydans HO-1, while dnfH, dnfG, and dnfD were not essential genes. Furthermore, an unexpected redistribution phenomenon for nitrogen between Dirammox and cell growth for ammonia removal in HO-1 was revealed. After the disruption of the Dirammox in HO-1, more consumed NH4 + was recovered as biomass-N via rapid metabolic response and upregulated expression of genes associated with ammonia transport and assimilation, tricarboxylic acid cycle, sulfur metabolism, ribosome synthesis, and other molecular functions. These findings deepen our understanding of the molecular mechanisms for Dirammox process in the genus Alcaligenes and provide useful information about the application of Alcaligenes species for ammonia-rich wastewater treatment.

Nonsterilized Fermentation of Crude Glycerol for Polyhydroxybutyrate Production by Metabolically Engineered Vibrio natriegens.

Polyhydroxybutyrate (PHB) is an attractive biodegradable polymer that can be produced through the microbial fermentation of organic wastes or wastewater. However, its mass production has been restricted by the poor utilization of organic wastes due to the presence of inhibitory substances, slow microbial growth, and high energy input required for feedstock sterilization. Here, Vibrio natriegens, a fast-growing bacterium with a broad substrate spectrum and high tolerance to salt and toxic substances, was genetically engineered to enable efficient PHB production from nonsterilized fermentation of organic wastes. The key genes encoding the PHB biosynthesis pathway of V. natriegens were identified through base editing and overexpressed. The metabolically engineered strain showed 166-fold higher PHB content (34.95 wt %) than the wide type when using glycerol as a substrate. Enhanced PHB production was also achieved when other sugars were used as feedstock. Importantly, it outperformed the engineered Escherichia coli MG1655 in PHB productivity (0.053 g/L/h) and tolerance to toxic substances in crude glycerol, without obvious activity decline under nonsterilized fermentation conditions. Our work demonstrates the great potential of engineered V. natriegens for low-cost PHB bioproduction and lays a foundation for exploiting this strain as a next-generation model chassis microorganism in synthetic biology.

Efficient and precise control of gene expression in Geobacter sulfurreducens through new genetic elements and tools for pollutant conversion.

Geobacter species, exhibiting exceptional extracellular electron transfer aptitude, hold great potential for applications in pollution remediation, bioenergy production, and natural elemental cycles. Nonetheless, a scarcity of well-characterized genetic elements and gene expression tools constrains the effective and precise fine-tuning of gene expression in Geobacter species, thereby limiting their applications. Here, we examined a suite of genetic elements and developed a new genetic editing tool in Geobacter sulfurreducens to enhance their pollutant conversion capacity. First, the performances of the widely used inducible promoters, constitutive promoters, and ribosomal binding sites (RBSs) elements in G. sulfurreducens were quantitatively evaluated. Also, six native promoters with superior expression levels than constitutive promoters were identified on the genome of G. sulfurreducens. Employing the characterized genetic elements, the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system was constructed in G. sulfurreducens to achieve the repression of an essential gene-aroK and morphogenic genes-ftsZ and mreB. Finally, applying the engineered strain to the reduction of tungsten trioxide (WO3 ), methyl orange (MO), and Cr(VI), We found that morphological elongation through ftsZ repression amplified the extracellular electron transfer proficiency of G. sulfurreducens and facilitated its contaminant transformation efficiency. These new systems provide rapid, versatile, and scalable tools poised to expedite advancements in Geobacter genomic engineering to favor environmental and other biotechnological applications.

Phthalates Promote Dissemination of Antibiotic Resistance Genes: An Overlooked Environmental Risk.

Plastics-microorganism interactions have aroused growing environmental and ecological concerns. However, previous studies concentrated mainly on the direct interactions and paid little attention to the ecotoxicology effects of phthalates (PAEs), a common plastic additive that is continuously released and accumulates in the environment. Here, we provide insights into the impacts of PAEs on the dissemination of antibiotic resistance genes (ARGs) among environmental microorganisms. Dimethyl phthalate (DMP, a model PAE) at environmentally relevant concentrations (2-50 µg/L) significantly boosted the plasmid-mediated conjugation transfer of ARGs among intrageneric, intergeneric, and wastewater microbiota by up to 3.82, 4.96, and 4.77 times, respectively. The experimental and molecular dynamics simulation results unveil a strong interaction between the DMP molecules and phosphatidylcholine bilayer of the cell membrane, which lowers the membrane lipid fluidity and increases the membrane permeability to favor transfer of ARGs. In addition, the increased reactive oxygen species generation and conjugation-associated gene overexpression under DMP stress also contribute to the increased gene transfer. This study provides fundamental knowledge of the PAE-bacteria interactions to broaden our understanding of the environmental and ecological risks of plastics, especially in niches with colonized microbes, and to guide the control of ARG environmental spreading.

Biomolecular Insights into Extracellular Pollutant Reduction Pathways of Geobacter sulfurreducens Using a Base Editor System.

Geobacter species are critically involved in elemental biogeochemical cycling and environmental bioremediation processes via extracellular electron transfer (EET), but the underlying biomolecular mechanisms remain elusive due to lack of effective analytical tools to explore into complicated EET networks. Here, a simple and highly efficient cytosine base editor was developed for engineering of the slow-growing Geobacter sulfurreducens (a doubling time of 5 h with acetate as the electron donor and fumarate as the electron acceptor). A single-plasmid cytosine base editor (pYYDT-BE) was constructed in G. sulfurreducens by fusing cytosine deaminase, Cas9 nickase, and a uracil glycosylase inhibitor. This system enabled single-locus editing at 100% efficiency and showed obvious preference at the cytosines in a TC, AC, or CC context than in a GC context. Gene inactivation tests confirmed that it could effectively edit 87.7-93.4% genes of the entire genome in nine model Geobacter species. With the aid of this base editor to construct a series of G. sulfurreducens mutants, we unveiled important roles of both pili and outer membrane c-type cytochromes in long-range EET, thereby providing important evidence to clarify the long-term controversy surrounding their specific roles. Furthermore, we find that pili were also involved in the extracellular reduction of uranium and clarified the key roles of the ExtHIJKL conduit complex and outer membrane c-type cytochromes in the selenite reduction process. This work developed an effective base editor tool for the genetic modification of Geobacter species and provided new insights into the EET network, which lay a basis for a better understanding and engineering of these microbes to favor environmental applications.

Repurposing CRISPR RNA-guided integrases system for one-step, efficient genomic integration of ultra-long DNA sequences.

Genomic integration techniques offer opportunities for generation of engineered microorganisms with improved or even entirely new functions but are currently limited by inability for efficient insertion of long genetic payloads due to multiplexing. Herein, using Shewanella oneidensis MR-1 as a model, we developed an optimized CRISPR-associated transposase from cyanobacteria Scytonema hofmanni (ShCAST system), which enables programmable, RNA-guided transposition of ultra-long DNA sequences (30 kb) onto bacterial chromosomes at ∼100% efficiency in a single orientation. In this system, a crRNA (CRISPR RNA) was used to target multicopy loci like insertion-sequence elements or combining I-SceI endonuclease, thereby allowing efficient single-step multiplexed or iterative DNA insertions. The engineered strain exhibited drastically improved substrate diversity and extracellular electron transfer ability, verifying the success of this system. Our work greatly expands the application range and flexibility of genetic engineering techniques and may be readily extended to other bacteria for better controlling various microbial processes.

Unexpected role of electron-transfer hub in direct degradation of pollutants by exoelectrogenic bacteria.

Exoelectrogenic bacteria (EEB) are capable of anaerobic respiration with diverse extracellular electron acceptors including insoluble minerals, electrodes and flavins, but the detailed electron transfer pathways and reaction mechanisms remain elusive. Here, we discover that CymA, which is usually considered to solely serve as an inner-membrane electron transfer hub in Shewanella oneidensis MR-1 (a model EEB), might also function as a reductase for direct reducing diverse nitroaromatic compounds (e.g. 2,4-dichloronitrobenzene) and azo dyes. Such a process can be accelerated by dosing anthraquinone-2,6-disulfonate. The CymA-based reduction pathways in S. oneidensis MR-1 for different contaminants could be functionally reconstructed and strengthened in Escherichia coli. The direct reduction of lowly polar contaminants by quinol oxidases like CymA homologues might be universal in diverse microbes. This work offers new insights into the pollutant reduction mechanisms of EEB and unveils a new function of CymA to act as a terminal reductase.

Engineering of salt-tolerant Shewanella aquimarina XMS-1 for enhanced pollutants transformation and electricity generation.

Saline wastewater poses a challenge during bio-treatment process due to salinity affecting the physiological activity of microorganisms and inhibiting their growth and metabolism. Thus, screening and engineering the salt-tolerant strains with stronger performances are urgent. Shewanella aquimarina XMS-1, a salt-tolerant dissimilated metal reducing bacteria (DMRB), was isolated from seawater environment. Its ability for reducing pollutants and generating electricity was enhanced by overexpression of riboflavin synthesis pathway encoding genes from S. oneidensis MR-1 under salt stress. Furthermore, upon contact with graphene oxide (GO), the engineered strain XMS-1/pYYDT-rib with enhanced flavins synthesis could reduce GO and self-assemble to form a three-dimensional (3D) biohybrid system named XMS-1/flavins/rGO. This 3D biohybrid system significantly enhanced the EET efficiency of S. aquimarina XMS-1. Our findings provide a feasible strategy for treatment of salt-containing industrial wastewater contaminated by metal and organic pollutants.

Extracellular electron transfer via multiple electron shuttles in waterborne Aeromonas hydrophila for bioreduction of pollutants.

Members of the genus Aeromonas prevail in aquatic habitats and have a great potential in biological wastewater treatment because of their unique extracellular electron transfer (EET) capabilities. However, the mediated EET mechanisms of Aeromonas have not been fully understood yet, hindering their applications in biological wastewater treatment processes. In this study, the electron shuttles in Aeromonas hydrophila, a model and widespread strain in aquatic environments and wastewater treatment plants, were explored. A. hydrophila was found to produce both flavins and 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ) as electron shuttles and utilize them to accelerate its EET for the bioreduction of various pollutants. The Mtr-like respiratory pathway was essential for the reduction of flavins, but not involved in the ACNQ reduction. The electron shuttle activity of ACNQ for pollutant bioreduction involved the redox reactions that occurred inside the cell. These findings deepen our understanding about the underlying EET mechanisms in dissimilatory metal reducing bacteria and provide new insights into the roles of the genus Aeromonas in biological wastewater treatment.

Unexpected Discovery of Hypermutator Phenotype Sounds the Alarm for Quality Control Strains.

Microbial strains with high genomic stability are particularly sought after for testing the quality of commercial microbiological products, such as biological media and antibiotics. Yet, using mutation-accumulation experiments and de novo assembled complete genomes based on Nanopore long-read sequencing, we find that the widely used quality-control strain Shewanella putrefaciens ATCC-8071, also a facultative pathogen, is a hypermutator, with a base-pair substitution mutation rate of 2.42 × 10-8 per nucleotide site per cell division, ∼146-fold greater than that of the wild-type strain CGMCC-1.6515. Using complementation experiments, we confirm that mutL dysfunction, which was a recent evolutionary event, is the cause for the high mutation rate of ATCC-8071. Further analyses also give insight into possible relationships between mutation and genome evolution in this important bacterium. This discovery of a well-known strain being a hypermutator necessitates screening the mutation rate of bacterial strains before any quality control or experiments.

Developing a base-editing system to expand the carbon source utilization spectra of Shewanella oneidensis MR-1 for enhanced pollutant degradation.

Shewanella oneidensis MR-1, a model strain of exoelectrogenic bacteria (EEB), plays a key role in environmental bioremediation and bioelectrochemical systems because of its unique respiration capacity. However, only a narrow range of substrates can be utilized by S. oneidensis MR-1 as carbon sources, resulting in its limited applications. In this study, a rapid, highly efficient, and easily manipulated base-editing system pCBEso was developed by fusing a Cas9 nickase (Cas9n (D10A)) with the cytidine deaminase rAPOBEC1 in S. oneidensis MR-1. The C-to-T conversion of suitable C within the base-editing window could be readily and efficiently achieved by the pCBEso system without requiring double-strand break or repair templates. Moreover, double-locus simultaneous editing was successfully accomplished with an efficiency of 87.5%. With this tool, the key genes involving in N-acetylglucosamine (GlcNAc) or glucose metabolism in S. oneidensis MR-1 were identified. Furthermore, an engineered strain with expanded carbon source utilization spectra was constructed and exhibited a higher degradation rate for multiple organic pollutants (i.e., azo dyes and organoarsenic compounds) than the wild-type when glucose or GlcNAc was used as the sole carbon source. Such a base-editing system could be readily applied to other EEB. This study not only enhances the substrate utilization and pollutant degradation capacities of S. oneidensis MR-1 but also accelerates the robust construction of engineered strains for environmental bioremediation.

CRISPRi System as an Efficient, Simple Platform for Rapid Identification of Genes Involved in Pollutant Transformation by Aeromonas hydrophila.

Aeromonas species are indigenous in diverse aquatic environments and play important roles in environmental remediation. However, the pollutant transformation mechanisms of these bacteria remain elusive, and their potential in pollution control is largely unexploited so far. In this work, we report an efficient and simple genome regulation tool to edit Aeromonas hydrophila and identify its biomolecular pathways for pollutant transformation. The genome regulation system, which is based on the type II clustered regularly interspaced short palindromic repeat interference (CRISPRi) system from Streptococcus pyogenes, can serve as a reversible and multiplexible platform for gene knockdown in A. hydrophila. A single-plasmid CRISPRi system harboring both dCas9 and the sgRNA was constructed in A. hydrophila and used to silence diverse genes with varied sizes and expression levels. With this system, up to 467-fold repression of gfp expression was achieved, and the function of the essential gene-ftsZ was identified quickly and accurately. Furthermore, simultaneous transcriptional repression of multiple targeted genes was realized. We discovered that the ars operon played an essential role in arsenic detoxification, and the extracellular electron transfer (EET) pathway was involved in methyl orange reduction, but not in vanadium reduction by A. hydrophila. Our method allows better insights and effective genetic manipulation of the pollutant transformation processes in Aeromonas, which might facilitate more efficient utilization of the Aeromonas species and other microbial species for environmental remediation applications.

Promoting bidirectional extracellular electron transfer of Shewanella oneidensis MR-1 for hexavalent chromium reduction via elevating intracellular cAMP level.

The bioreduction capacity of Cr(VI) by Shewanella is mainly governed by its bidirectional extracellular electron transfer (EET). However, the low bidirectional EET efficiency restricts its wider applications in remediation of the environments contaminated by Cr(VI). Cyclic adenosine 3',5'-monophosphate (cAMP) commonly exists in Shewanella strains and cAMP-cyclic adenosine 3',5'-monophosphate receptor protein (CRP) system regulates multiple bidirectional EET-related pathways. This inspires us to strengthen the bidirectional EET through elevating the intracellular cAMP level in Shewanella strains. In this study, an exogenous gene encoding adenylate cyclase from the soil bacterium Beggiatoa sp. PS is functionally expressed in Shewanella oneidensis MR-1 (the strain MR-1/pbPAC) and a MR-1 mutant lacking all endogenous adenylate cyclase encoding genes (the strain Δca/pbPAC). The engineered strains exhibit the enhanced bidirectional EET capacities in microbial electrochemical systems compared with their counterparts. Meanwhile, a three times more rapid reduction rate of Cr(VI) is achieved by the strain MR-1/pbPAC than the control in batch experiments. Furthermore, a higher Cr(VI) reduction efficiency is also achieved by the strain MR-1/pbPAC in the Cr(VI)-reducing biocathode experiments. Such a bidirectional enhancement is attributed to the improved production of cAMP-CRP complex, which upregulates the expression levels of the genes encoding the c-type cytochromes and flavins synthetic pathways. Specially, this strategy could be used as a broad-spectrum approach for the other Shewanella strains. Our results demonstrate that elevating the intracellular cAMP levels could be an efficient strategy to enhance the bidirectional EET of Shewanella strains and improve their pollutant transformation capacity.